CN110923243B

CN110923243B - Application of AHL4 in regulation and control of plant lipid metabolism and method for increasing oil content and unsaturated fatty acid content of plant seeds

Info

Publication number: CN110923243B
Application number: CN201911310131.9A
Authority: CN
Inventors: 蔡光勤; 王学敏; 周永明
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-12-18
Filing date: 2019-12-18
Publication date: 2021-04-13
Anticipated expiration: 2039-12-18
Also published as: CN110923243A

Abstract

The invention belongs to the technical field of crop genetic breeding, and particularly relates to application of AHL4 in regulation and control of plant lipid metabolism and a method for improving oil content and unsaturated fatty acid content of plant seeds. The research of the invention finds that AHL4 can be specifically combined with unsaturated phosphatidic acid, and finds that AHL4 can regulate and control the establishment of a plant seedling system; it is presumed that the effect of AHL4 gene on establishment of young plant system is probably achieved by regulation of lipid metabolism. By comparing the rates of TAG hydrolysis of AHL4 mutants and over-expressed plant seeds, it was first discovered and confirmed that AHL4 transcription factor is indeed able to influence plant lipid metabolism, and this conclusion was validated from gene expression levels, providing mechanistic support at the gene level. The invention obviously improves the contents of grease and unsaturated fatty acid in plant seeds by over-expressing AHL4 gene in plants.

Description

Application of AHL4 in regulation and control of plant lipid metabolism and method for increasing oil content and unsaturated fatty acid content of plant seeds

Technical Field

The invention belongs to the technical field of crop genetic breeding, and particularly relates to application of AHL4 in regulation and control of plant lipid metabolism and a method for improving oil content and unsaturated fatty acid content of plant seeds.

Background

Plant seed germination (germination) and the subsequent establishment of seedlings (seedling) are crucial for the whole life cycle of a plant.

Lipids (lipids), mainly in the form of Triacylglycerols (TAGs), are stored in plant mature seeds. The lipids in mature seeds are the most dominant source of energy for plant seed germination and later seedling establishment before the plant is able to perform photosynthesis.

The degradation of oil in plants is regulated by complex biological processes, and the currently known biological mechanisms mainly existing in the degradation of oil in plants include the following processes: (1) the grease degrades TAG into DAG (diacylglycerol) and MAG (monoacylglycerol) under the action of triacylglycerol lipase (TAG lipase), and then free fatty acid (free fatty acid) is released; (2) free fatty acids transport the fatty acids produced by hydrolysis into peroxisomes (peroxisomes) by the action of a transporter (transporter); (3) in peroxisomes, fatty acids undergo multiple rounds of complex β -oxidation, each round of β -oxidative degradation producing acetyl-CoA (acetyl-CoA) until complete degradation of the fatty acid; beta-oxidation is primarily regulated by four classes of genes in higher plants: long-chain acyl-CoA synthases (LACS), acyl-CoA oxidases (ACXs), multifunctional proteins (MFPs), and 3-keto acyl-CoA thiolases (KATs); (4) the acetyl coenzyme A generated by degradation is transported to glyoxylate bodies (glyxosomes) for glyoxylate cycle, and finally glucose (glucose) is generated for plant seed germination and seedling stage establishment. Glucose produced by lipid degradation of plant seeds themselves is a major source of energy for plant seed germination and seedling stage establishment before the plants themselves are able to perform photosynthesis. Therefore, the regulation of lipid metabolism in the seedling establishment period of seeds is a powerful means for regulating the efficient growth of plants.

All genes known to date in plants which are involved in lipid degradation are various enzymes (enzymes), but the biological regulation of the lipid degradation process is not known at present.

Phosphatidic Acid (PA) plays a crucial role as a signal molecule in plant cells in lipid-related biological regulation. Transcription Factor (TF), as a gene regulating the expression of downstream genes, plays an important role in the regulation of plant biological processes.

AHL (AT-hook motif) -like transcription factor is a DNA binding transcription factor specific to plants and prokaryotes, two conserved structural domains exist in the transcription factor, one is DNA binding AT-hook (DNA binding AT-hook motif), and the transcription factor has a conserved amino acid sequence Gly-Arg-Pro and can regulate the chromosome structure so as to regulate transcription; the other is the 296 unknown functional domain (unknown function #296(DUF296)), annotated as a conserved domain in plants and prokaryotes (plant and prokaryote conserved (PPC) domain). There are 29 AHLs in arabidopsis that vary in function and are involved in plant growth and development (e.g., vascular tissue development, hypocotyl elongation, development of leaves and flowers, leaf senescence, growth period, etc.), stress response (e.g., plant innate immunity, etc.), and the like. However, no reports related to lipid regulation exist.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an application of AHL4 in regulation and control of plant lipid metabolism and a method for increasing the oil content and unsaturated fatty acid content of plant seeds, and aims to solve part of the problems in the prior art or at least alleviate part of the problems in the prior art.

The invention is realized by the application of the AHL4 transcription factor in regulating and controlling the metabolism of plant lipid.

Further, the application shows that AHL4 gene deletion promotes TAG hydrolysis; overexpression of the AHL4 gene inhibited TAG hydrolysis.

Further, AHL4 gene deletion promotes TAG hydrolysis by promoting TAG hydrolysis and expression of β -oxidation related genes; AHL4 gene overexpression achieves inhibition of TAG hydrolysis by inhibiting expression of TAG hydrolysis and β -oxidation related genes.

Further, the TAG hydrolysis related genes include SDP1, DALL 5; the beta-oxidation related gene comprises KAT 5.

Application of AHL4 transcription factor in regulating plant seedling system establishment.

Further, the application shows that AHL4 gene deletion promotes germination rate and/or main root elongation of plant seeds; AHL4 gene overexpression inhibits the germination rate and/or main root elongation of plant seeds.

A method of increasing the oil content of a plant seed comprising overexpressing an AHL4 gene in the plant.

Further, the method for over-expressing the AHL4 gene in the plant comprises the following steps: the primer sequences shown in SEQ ID NO.5 and SEQ ID NO.6 are utilized to amplify in an arabidopsis thaliana wild type DNA template to obtain a target fragment, then the target fragment is connected to a pBin35sRed1 vector in an enzyme digestion manner, and then the recombinant plasmid is converted into the arabidopsis thaliana wild type by adopting an agrobacterium dipping method.

A method of increasing the unsaturated fatty acid content of a plant seed comprising overexpressing an AHL4 gene in the plant.

In summary, the advantages and positive effects of the invention are:

according to the research of the invention, AHL4 can be specifically combined with unsaturated Phosphatidic Acid (PA), and the comparison of different expression effects of an AHL4 knockout mutant and an overexpression plant on the establishment of a plant seedling stage shows that the AHL4 transcription factor can regulate the establishment of a plant seedling system, and specifically shows that AHL4 gene deletion promotes the germination rate and/or main root elongation of plant seeds; AHL4 gene overexpression inhibits the germination rate and/or main root elongation of plant seeds; it is presumed that the effect of AHL4 gene on establishment of young plant system is probably achieved by regulation of lipid metabolism.

Further, by comparing the rates of TAG hydrolysis of AHL4 mutants and over-expressed plant seeds, AHL4 transcription factors were first discovered and confirmed to be indeed able to influence plant lipid metabolism, and further validated this conclusion from gene expression levels and provided mechanistic support at the gene level.

Finally, the invention obviously improves the contents of grease and unsaturated fatty acid in plant seeds by over-expressing AHL4 gene in the plant.

The invention has the advantages and positive effects that: AHL4 is the first identified transcription factor currently known to regulate lipid degradation; 2. the oil content of the plant seeds can be obviously improved by over-expressing AHL 4; 3. the 8.6% oleic and linoleic acid content of plant seeds can be increased by overexpressing AHL 4.

Drawings

FIG. 1 shows that AHL4 specifically binds to Phosphatidic Acid (PA); AHL4 and different phospholipids and galactolipids (left), and different components of phosphatidic acid (right) combined, chloroform (Chl) as a negative control; b: detecting the combination condition of AHL4 and phosphatidic acid of different lipids and different components by utilizing liposome combination; c: analyzing the combination condition of AHL4 and phosphatidic acid by using surface plasmon resonance; d: overexpresses AHL4 in the plant body, and detects the combination of AHL4 and phosphatidic acid and components thereof in the plant body;

FIG. 2 is the effect of AHL4 knockout and overexpression plants during seed germination and seedling stage establishment; a: schematic diagram of insertion sites of 3 AHL 4T-DNA insertion mutants; b: PCR identification and semi-quantitative PCR results of three AHL 4T-DNA insertion homozygous mutants; c: overexpressing AHL4 in plants and detecting the overexpressed protein; d: AHL4 mutant, complementation, overexpression and wild type seeds germination rate statistics on sugarless (left) and 1% sugar (right) media; e: major root length statistics on no-sugar (left) and 1% sugar (right) media for AHL4 mutant, complementation line, overexpression line, and wild type seed;

FIG. 3 shows the Triacylglycerol (TAG) content and Phosphatidic Acid (PA) content during establishment of the seedling stage of AHL4 mutant and overexpressing plants; a: comparison of TAG degradation rates of AHL4 mutant, overexpression line, and wild type seeds on sugarless (left) and 1% sugar (right) media; b: measurement of phosphatidic acid content of AHL4 mutant, overexpression line and wild type seed on sugarless (left) and 1% sugar (right) culture medium at different periods; c: measurement of expression pattern of AHL4 gene on sugarless and 1% sugar medium;

FIG. 4 shows the change in expression levels of TAG hydrolysis and β -oxidation related genes 1 day after germination of AHL4 mutant and overexpressed plants on sugar-free (upper panel) and sugar-containing medium (lower panel);

FIG. 5 shows AHL4 binding to specific regions of the promoter of SDP1, DALL5 and KAT 5; a: gel retardation detection of AHL4 and different DNA fragment binding conditions, LHY gene as control; b: chromatin immunoprecipitation to detect binding of AHL4 to target DNA;

FIG. 6 is a graph showing that PA inhibits the binding of AHL4 to DNA;

FIG. 7 is a graph of the change in oil content (left) and fatty acid composition (right) of AHL4 mutant and over-expressed material seeds;

figure 8 is a diagram of the mode of operation of AHL4 in plants.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

The proteins or fragments thereof involved in the present invention may be recombinant, natural, synthetic proteins or fragments thereof; the proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant techniques.

Example 1AHL4 specifically binds to unsaturated Phosphatidic Acid (PA)

In the embodiment, AHL4 protein is specifically expressed in Escherichia coli, and purified by His antibody to obtain purified AHL4 protein. The binding of AHL4 to the different lipids was then analyzed using the membrane hybridization method (Filter-blotting assay).

1. Specific expression of AHL4 protein in Escherichia coli

In this example, AHL4 full-length CDS was cloned by high fidelity enzyme in a wild type Arabidopsis thaliana (Col-0) cDNA library using the following sequence primer pairs. The Arabidopsis AHL4 CDS sequence is shown in SEQ ID NO. 1.

AHL4-F：

5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGAGGAGAGAGAAGGAACT AACATCAA-3', see SEQ ID NO. 3;

AHL4-R：

5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTGCTTGGAACCTCGGTGTCAGATTCGCT ATG-3', see SEQ ID NO. 4.

PCR program and system methods and systems reference the method of Expression of Brassica napus TTG2, a regulator of a chromium degree, and initiation plant sensitivity to salt stress by compressing the Expression of an axin biochemical gene, Li et al (2015), with PCR conditions: 30s at 98 ℃; 5s at 98 ℃; 10s at 60 ℃, 30s at 72 ℃ and 28 cycles; extension at 72 ℃ for 10 min. The PCR product was then cloned into pDONR221^TMTransferring the target product into pET-53-DEST through BP reaction in the carrier^TMIn a carrier (both the carrier and the reagent are available from commercial companies). The obtained vector was then transformed into the E.coli DE3 strain. Culturing the Escherichia coli to A₆₀₀When the OD value was about 0.4, IPTG (isopropyl. beta. -D-thiogalactopyranoside) was added to the final concentration of 0.1 mM. The culture was continued at room temperature for 6 hours, and the cultured E.coli was centrifuged to obtain a product. Then lysis was performed with lysis buffer (50mM NaH)₂PO₄ pExtracting total protein with H8.0, 300mM NaCl, 0.1% (v/v) Tween-20, 1mM PMSF, purifying the total protein with Ni-NTA Agarose resin (available from commercial company), and obtaining AHL4 protein after purification.

2. Protein immunoblotting

Loading 10mg of target protein onto 10% SDS-PAGE gel until the protein is completely separated (about 2 hours), and transferring the protein on the gel onto a PVDF (polyvinylidene fluoride) membrane by an electrotransfer instrument; after incubating the membrane in 5% skim milk for 1 hour, the membrane is transferred to a primary antibody (e.g., anti-His antibody, available from commercial companies) for 1 hour, then transferred to a secondary antibody (e.g., anti-mouse antibody, available from commercial companies) for 1 hour, and finally subjected to target protein development using an ap (alkaline Phosphatase Conjugate substrate) development system (available from commercial companies).

3. Lipid-protein membrane hybridization

50 μ g of chloroform-soluble lipids (the types of lipids include PC, PE, PG, PS, MGDG, DGDG, and PA of its various components, all available from commercial companies) were dropped onto a cut nitrocellulose membrane (0.45 m), and after thorough drying, the membrane was incubated in 1% defatted BSA (fat acid free-bone serum albumin) for 1 hour, then the membrane was transferred to primary antibody for 1 hour, then secondary antibody for 1 hour, and finally developed with AP.

The results are shown in FIG. 1: AHL4 binds specifically only to PA, but not to PC (phosphatidylcholine), PE (phosphatidylethanolamine), PG (phosphatidylglycerol), PI (phosphatidylinositol), PS (phosphatidylserine), MGDG (galactosylglyceride), DGDG (digalactosyldiacylglycerol), and the like. AHL4 was then found to specifically bind unsaturated phosphatidic acids, such as 18:1-18:1PA, 18:2-18:2PA, 16:0-18:1PA and 16:0-18:2PA, by binding assays for the different phosphatidic acid components.

4. The method of liposome binding assay is used for further verifying that AHL4 can only specifically bind to unsaturated phosphatidic acid. The specific method comprises the following steps:

phosphatidylcholine (PC) and other lipids (e.g. different fractions of PA, PG, PE, MGDG, DGDG etc.) were mixed in a molar ratio of 3:1, and finally 0.8. mu. mol was dried with nitrogen and dissolved by adding HBS buffer (20mM HEPES pH7.5,100mM NaCl, and 0.02% (w/v) sodium azide), the solution was sonicated for 1 minute, then centrifuged at 4 degrees 50,000g for 20 minutes, the supernatant was discarded, the pellet was dissolved with 1ml binding buffer (25mM Tris-HCl pH7.5,125mM KCl,1mM DTT, and 0.5mM EDTA), then 10. mu.g purified AHL4 protein was added and incubated at room temperature for 1 hour. After incubation, the cells were centrifuged at 4 degrees at 16,000g for 30 minutes, washed 3 times, and subjected to SDS-PAGE.

5. The ability of AHL4 to bind to PA was further demonstrated using a surface plasmon resonance analysis (SPR) analysis. The specific method comprises the following steps:

PC and PA were mixed at a molar ratio of 3:1, then dissolved with running buffer (10mM HEPES pH7.4,0.15M NaCl,0.05mM EDTA) and analyzed using Biacore 2000system, all procedures being performed according to the instructions.

6. This example transiently expresses AHL4 in arabidopsis thaliana, followed by purification of the AHL4 protein complex by His tag, separation of lipids above the complex, followed by lipidomics analysis using mass spectrometry.

The over-expressed material was prepared by the following method: and amplifying in the arabidopsis wild-type DNA by using a primer pair to obtain a target fragment. PCR program and system methods and systems reference the method of Expression of Brassica napus TTG2, a regulator of a chromium degree, and initiation plant sensitivity to salt stress by compressing the Expression of an axin biochemical gene, Li et al (2015), with PCR conditions: 30s at 98 ℃; 5s at 98 ℃; 10s at 60 ℃, 120s at 72 ℃ and 28 cycles; extension at 72 ℃ for 10 min. Then the target fragment is cut and connected into a pBin35sRed1 vector by enzyme, and the plasmid is transformed into an arabidopsis wild type by adopting an agrobacterium flower dipping method. The amino acid sequence of Arabidopsis AHL4 is shown in SEQ ID NO.7, and the genome sequence encoding the amino acid in this example is shown in SEQ ID NO. 2. The PCR amplification primer sequences are as follows: AHL 4-F-XbaI:

5'-GCTCTAGAATGGAGGAGAGAGAAGGAACTA-3', see SEQ ID NO. 5;

AHL4-R-SmaI：

5'-TCCCCCGGGTCAAGCGTAATCTGGAACATCGTATGGGTAGCTTGGAACCTCGGTGTC AGA-3', see SEQ ID NO. 6.

The results show that high abundance of PA was detected in the over-expressed plants, further demonstrating that AHL4 binds to PA in plants.

Example 2 AHL4 knockout mutants and overexpressing plants show different effects on plant seedling establishment

To further investigate the function of AHL4 in plants, 3 independent T-DNA insertion mutants (knockout mutants) of AHL4 were purchased at the SALK institute in this example, while overexpressing the gene in arabidopsis thaliana with reference to the method in example 1, and a high abundance of AHL4 protein was detected using western blot.

In this example, the above materials were sown on 1/2MS sugar-free and 1% sucrose-containing medium, and the germination rate of the seeds was measured, as shown in FIG. 2D and FIG. 2E, wherein the left panels of FIGS. 2D and E are data results on 1/2MS sugar-free medium; FIGS. 2D and E, right panels, show the results of data on 1% sucrose medium.

The results show that AHL4 mutants (AHL4-1 and AHL4-2) have a significantly faster germination rate than the wild type on both sugar-free and sugar-containing media, whereas the complementary material did not differ from the wild type. However, the germination rate of AHL4 over-expressed material (OE-1, OE-2) was significantly slower than that of wild type. For example, at 10 hours, AHL4 mutant germinated 13% more than the wild type, whereas the over-expressed material was 18% less than the wild type. After the seeds germinate, radicles are exposed and then main roots develop, and the measurement of the main root length of the materials shows that the main root length of the AHL4 mutant is longer than that of the wild type on a culture medium which does not contain sugar and contains sugar and is 2-4 days after the seeds germinate, and the complementary line and the wild type have no difference; however, the taproots of AHL4 over-expressed material were barely elongated on sugar-free medium. For example, 3 days after germination, the AHL4 mutant had 34% longer main roots than the wild type, whereas the over-expressed material was 63% lower than the wild type and hardly elongated. This suggests that plants cannot establish their seedling system after overexpression of AHL4, which is likely to be associated with lipolysis (failing to provide relevant nutrients for plant seed germination and later seedling establishment).

Example 3 AHL4 overexpressing plants were unable to efficiently hydrolyze Triacylglycerols (TAGs) during the seedling stage establishment phase

To further verify whether AHL4 affects TAG hydrolysis, changes in TAG and PA content at different times on sugar-free and sugar-containing media were measured for AHL4 mutants and over-expressed plant seeds in this example. Methods for the detection of TAG and PA content refer to the method of Overexpression of a protein-related phospholipases AIII delta altered plant growth and engineered seed oil content in camelina. Li et al (2015).

The results are shown in FIG. 3, where the left panels in FIGS. 3A and B are the test data on the sugar-free medium; FIGS. 3A and B are right panels showing the data detected on the sugar-containing medium. The results show that the AHL4 mutant hydrolyzed TAG significantly faster than the wild type on both sugar-containing and sugar-free media, while the over-expressed strain was significantly slower than the wild type, especially on sugar-free media. For example, on day three post-germination, the AHL4 mutant degraded TAG 33% faster than the wild type on sugar-free medium, whereas AHL4 overexpressing line was 64% slower than the wild type, with little degradation. This further illustrates that AHL4 correlates with TAG hydrolysis regulation during plant seedling establishment. Measurement of PA content showed that the AHL4 mutant had higher PA content than wild type at different seedling establishment stages on both sugar-free and sugar-containing media, whereas the over-expressed plants were lower than wild type. For example, the AHL4 mutant has a PA content 14% higher than the wild type on sugar-free medium, while the AHL4 overexpression line is 52% lower than the wild type on the third day after germination. Further elucidating that PA is also involved in TAG hydrolysis during the seedling establishment phase.

Example 4 AHL4 Effect on TAG hydrolysis and expression of beta-Oxidation related genes

In order to further verify that AHL4 regulates TAG hydrolysis and beta-oxidation during plant seedling establishment, the change of TAG hydrolysis and beta-oxidation related gene expression level during key plant seedling establishment periods of AHL4 mutant and over-expressed plants is detected by a fluorescent quantitative PCR method in the embodiment.

By analysis of the expression pattern of AHL4 at the establishment stage of the seedling stage (see fig. 3C), it was established that 1 day after germination was the critical stage. Therefore, in this example, RNA was extracted from the above-mentioned material 1 day after germination on a medium containing no sugar and a medium containing sugar, and the expression level of the relevant gene was analyzed. The method and system for RNA extraction, reverse transcription and real-time PCR of plant tissues refer to the method of Expression of Brassica napus TTG2, a regulator of a chromosome level, and Expression of Expression by Expression of an axin biochemical genes Li et al (2015).

The results are shown in FIG. 4, where the upper panel of FIG. 4 is the effect on a sugar-free medium; FIG. 4 the lower graph shows the effect on a sugar-containing medium. The results show that: of the genes involved in TAG hydrolysis and beta-oxidation, most of the genes were highly expressed in the mutants in the sugar-free and sugar-containing medium, while the expression was suppressed in the over-expressed material, especially the TAG hydrolase genes SDP1, DALL5 and beta-oxydrolytic enzyme gene KAT 5. For example, 1 day after germination, the AHL4 mutant expressed 2.3, 2.2, 3.2 times higher than the wild type in TAG hydrolase genes SDP1, DALL5, and β -oxydrolytic enzyme gene KAT5, respectively, on a sugar-free medium, while the AHL4 overexpression lines were 0.2, 0.4, 0.3 times higher than the wild type, respectively; the expression levels of the AHL4 mutant on a sugar-containing medium for the TAG hydrolase genes SDP1, DALL5 and beta-oxydrolytic enzyme gene KAT5 are respectively 1.3, 1.7 and 1.9 times of that of the wild type, while the expression levels of the AHL4 overexpression lines are respectively 0.4, 0.4 and 0.4 times of that of the wild type; it was further shown that AHL4 inhibits the expression of these genes and in turn affects the degradation of TAG.

Example 5 binding of AHL4 to specific regions of the promoters of SDP1, DALL5 and KAT5

To further verify that AHL4 regulates key genes in the above-mentioned pathway, in this example, whether AHL4 binds to a promoter specific region of the above-mentioned genes was investigated in vitro by a gel blocking method (EMSA). The specific method comprises the following steps:

the DNA probe comprises specific AT-rich fragments of SDP1, DALL5 and KAT5 gene promoter (detailed information shown in Table 1 below), and a fluorescent group (6-FAM) is added to the 5' end of the probe during synthesis. Mu.g of purified AHL4 protein was mixed with the DNA probes described above and other components (e.g., PC, PA, etc.), incubated at room temperature for 30 minutes, then electrophoresed using 2.5% agarose gel for 30 minutes, and the gel was imaged under the Azure C600 imager system.

Table 1: primer and Probe information used in the experiment (5 'to 3')

The results are shown in FIG. 5 and show that AHL4 can bind to AT base rich (AT rich) regions on the promoters of SDP1, DALL5 and KAT 5.

Thereafter, in this example, it was further verified whether AHL4 binds to a promoter specific region of the above gene in plants by extracting chromatin (chromatin) of AHL4 overexpressed and wild-type plants, and then immunoprecipitating an AHL4 protein complex (ChIP), using the method of ChIP-PCR. The results show that AHL4 can bind to AT base rich (AT rich) regions on the promoters of SDP1, DALL5 and KAT 5.

Example 6 PA is able to inhibit binding of AHL4 to DNA

Since AHL4 is a transcription factor for PA binding, in order to further confirm the role of PA in the transcriptional regulation of AHL4, the role of PA in binding of AHL4 and DNA was confirmed using the gel blocking (EMSA) method in this example.

The results are shown in FIG. 6, which shows: PA and unsaturated PA are added in the reaction, so that the combination of AHL4 and a specific DNA fragment can be inhibited; phosphatidylcholine and saturated PA did not inhibit binding of AHL4 to DNA.

Example 7 AHL4 is able to affect seed oil content and fatty acid composition

To further enrich the role of AHL4 in vegetable oil metabolism, the oil content and fatty acid composition of mature plant seeds were measured in this example. Methods for determining oil content and fatty acid composition are described in the methods of expression of protein-related phospholipases AIII delta altered plant growth and engineered oil content in camelina. Li et al. (2015).

The results are shown in fig. 7, and the results show that the oil content of the seeds of the plants over-expressed by AHL4 is significantly higher than that of the wild type, which indicates that AHL4 can inhibit the degradation of TAG during the oil accumulation process of the seeds of the plants, thereby increasing the oil content. Meanwhile, the content of unsaturated fatty acid in the seeds of the AHL4 overexpression plants is obviously higher than that of the wild plants, and is probably related to the fact that AHL4 can specifically bind to unsaturated PA. In the embodiment, the oil content of the plant seeds can be obviously improved by over-expressing AHL 4; can increase the content of oleic acid and linoleic acid of 8.6 percent in plant seeds.

Combining the above research results, the working mode of AHL4 in plant is shown in FIG. 8. AHL4 was able to specifically bind to the promoter specific regions of TAG lipase and β -oxidation related genes, while PA was able to inhibit the binding of AHL4 to DNA, thus creating a competitive relationship in plant cells. AHL4 did not inhibit the expression of TAG lipase and beta-oxidation related genes when AHL4 was deleted, thereby promoting seed germination and seedling establishment, while AHL4 was overexpressed in plants, which did not have sufficient PA to inhibit the binding of AHL4 to DNA, thereby inhibiting the expression of TAG lipase and beta-oxidation related genes, thereby affecting the seed germination rate and seedling establishment

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1260

<212> DNA

<213> nucleotide sequence (AHL4 CDS)

<400> 1

atggaggaga gagaaggaac taacatcaac aacatcccaa ccagttttgg tctgaaacaa 60

catgaaactc ctcttcctcc tcctggttac ccaccacggt ctgaaaaccc taatcttttt 120

ccggtgggtc aatccagcac ttcctccgcc gccgccgcgg tgaaaccttc tgagaatgtt 180

gctcctcctt ttagcttaac aatgccggtg gagaattctt cttctgagtt gaagaagaag 240

agagggagac caagaaagta taaccctgac ggctcactcg ctgtgactct ctctcctatg 300

cctatctcat cctccgttcc gttgacgtcg gagtttggtt ctcggaaacg aggaagaggt 360

cgaggaagag gcagaggaag aggacgagga cgtggacaag gacaaggaag cagagagccc 420

aataacaaca acaacgacaa caattggctc aagaatcctc agatgttcga atttaacaac 480

aacactccta cttctggtgg aggaggacct gctgaaattg tcagtccaag ttttacacct 540

catgtgctca cagtaaatgc cggtgaggat gtgacaatga agataatgac attctctcaa 600

caaggctcgc gtgctatttg tattctttca gcgaacggtc ccatatccaa tgttacactt 660

cgtcaatcta tgacatctgg tggtactctc acttatgagg gtcattttga gattctttct 720

ttgacgggtt cgtttatacc aagcgagagt ggaggaaccc gaagcagagc tggtgggatg 780

agtgtctctc ttgcaggaca agatggtcgt gtctttggtg gtggacttgc tggtctcttt 840

attgccgctg gtcctgttca ggtaatggta gggagtttta tagcgggtca ggaggaatcg 900

cagcagcagc agcagcagat aaagaagcaa agaagggaaa gactcgggat cccgacaaca 960

acacaagctt ctaatatctc attcggtggc tcagcggaag atcctaaggc tagatacggg 1020

ctcaacaagc ctgttgttat tcagccacca ccggtgtctg caccacctgt gtccttttcg 1080

catgaaccaa gtactaacac cgtccatggt tactatgcaa ataacacagc taaccatatc 1140

aaggatctct tctcttccct ccctggagaa gatagggaag aagatgagga tgatttagaa 1200

ggtgaagatg atgaagaatt cggaggccat agcgaatctg acaccgaggt tccaagctga 1260

<210> 2

<211> 2067

<212> DNA

<213> nucleotide sequence (AHL4)

<400> 2

atggaggaga gagaaggaac taacatcaac aacatcccaa ccagttttgg tctgaaacaa 60

catgaaactc ctcttcctcc tcctggttac ccaccacggt ctgaaaaccc taatcttttt 120

ccggtgggtc aatccagcac ttcctccgcc gccgccgcgg tgaaaccttc tgagaatgtt 180

gctcctcctt ttagcttaac aatgccggtg gagaattctt cttctgagtt gaagaagaag 240

agagggagac caagaaagta taaccctgac ggctcactcg ctgtgactct ctctcctatg 300

cctatctcat cctccgttcc gttgacgtcg gagtttggtt ctcggaaacg aggaagaggt 360

cgaggaagag gcagaggaag aggacgagga cgtggacaag gacaaggaag cagagagccc 420

aataacaaca acaacgacaa caattggctc aagaatcctc agatgttcga atttaacaac 480

aacactccta cttctggtaa tgtctttttc tcctttcctg ttcgaattta ggaatctctg 540

tgtgtgtaca ttggcctctc tgtataagaa tcaatagata gcttttaata gatctatttg 600

acttgcaaaa attttggaat aaggacaaag gttttgtctt ataaagctag tgaatgttac 660

tgtccagtaa actggaggat tctgtgtgaa aaaagagttg aatttaaagg aaaagtccca 720

gaagaagaag gcaatttagg gaaactagtt cggattcttt gatgatagtg acataagcag 780

tgtgattaag agtatatata tcgaggaatc tgtaggcttt ggtcttatta cttaaggatt 840

taacttgaac tctttactgt tttggtttta gcaatttgtt agttttcttg atcagatttc 900

gtttgtttta tctgttaagc ttgattcatt tgatgattaa tttgcaggtg gaggaggacc 960

tgctgaaatt gtcagtccaa gttttacacc tcatgtgctc acagtaaatg ccggtgaggt 1020

atgttttcga taattctatc tacttgtgat ttatttactg aattatttgt gagtataaag 1080

tcagtttagc gaccttaata ggaacatact atgattaacc aagctcatta ttcttgacta 1140

agtccacaag atctctaact atttttggcc cactgaatct aacaggatgt gacaatgaag 1200

ataatgacat tctctcaaca aggctcgcgt gctatttgta ttctttcagc gaacggtccc 1260

atatccaatg ttacacttcg tcaatctatg acatctggtg gtactctcac ttatgaggtt 1320

tgctctctct cttcactctc tcttagattc tttctaatct tttttctttt ttactaaatt 1380

tagtctacaa tttgtggtca gggtcatttt gagattcttt ctttgacggg ttcgtttata 1440

ccaagcgaga gtggaggaac ccgaagcaga gctggtggga tgagtgtctc tcttgcagga 1500

caagatggtc gtgtctttgg tggtggactt gctggtctct ttattgccgc tggtcctgtt 1560

caggtaataa cttagaccat gaggtgttgt gttgatctct ttaatggact cgtgtttttg 1620

tatcgtttta agcattgtgg ttttgaatac gatatgtata ttgttcaggt aatggtaggg 1680

agttttatag cgggtcagga ggaatcgcag cagcagcagc agcagataaa gaagcaaaga 1740

agggaaagac tcgggatccc gacaacaaca caagcttcta atatctcatt cggtggctca 1800

gcggaagatc ctaaggctag atacgggctc aacaagcctg ttgttattca gccaccaccg 1860

gtgtctgcac cacctgtgtc cttttcgcat gaaccaagta ctaacaccgt ccatggttac 1920

tatgcaaata acacagctaa ccatatcaag gatctcttct cttccctccc tggagaagat 1980

agggaagaag atgaggatga tttagaaggt gaagatgatg aagaattcgg aggccatagc 2040

gaatctgaca ccgaggttcc aagctga 2067

<210> 3

<211> 60

<212> DNA

<213> Artificial sequence (AHL4-F)

<400> 3

ggggacaagt ttgtacaaaa aagcaggctt catggaggag agagaaggaa ctaacatcaa 60

<210> 4

<211> 60

<212> DNA

<213> Artificial sequence (AHL4-R)

<400> 4

ggggaccact ttgtacaaga aagctgggtt gcttggaacc tcggtgtcag attcgctatg 60

<210> 5

<211> 30

<212> DNA

<213> Artificial sequence (AHL4-F-XbaI)

<400> 5

gctctagaat ggaggagaga gaaggaacta 30

<210> 6

<211> 60

<212> DNA

<213> Artificial sequence (AHL4-R-SmaI)

<400> 6

tcccccgggt caagcgtaat ctggaacatc gtatgggtag cttggaacct cggtgtcaga 60

<210> 7

<211> 419

<212> PRT

<213> amino acid sequence (AHL4)

<400> 7

Met Glu Glu Arg Glu Gly Thr Asn Ile Asn Asn Ile Pro Thr Ser Phe

1 5 10 15

Gly Leu Lys Gln His Glu Thr Pro Leu Pro Pro Pro Gly Tyr Pro Pro

20 25 30

Arg Ser Glu Asn Pro Asn Leu Phe Pro Val Gly Gln Ser Ser Thr Ser

35 40 45

Ser Ala Ala Ala Ala Val Lys Pro Ser Glu Asn Val Ala Pro Pro Phe

50 55 60

Ser Leu Thr Met Pro Val Glu Asn Ser Ser Ser Glu Leu Lys Lys Lys

65 70 75 80

Arg Gly Arg Pro Arg Lys Tyr Asn Pro Asp Gly Ser Leu Ala Val Thr

85 90 95

Leu Ser Pro Met Pro Ile Ser Ser Ser Val Pro Leu Thr Ser Glu Phe

100 105 110

Gly Ser Arg Lys Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly

115 120 125

Arg Gly Arg Gly Gln Gly Gln Gly Ser Arg Glu Pro Asn Asn Asn Asn

130 135 140

Asn Asp Asn Asn Trp Leu Lys Asn Pro Gln Met Phe Glu Phe Asn Asn

145 150 155 160

Asn Thr Pro Thr Ser Gly Gly Gly Gly Pro Ala Glu Ile Val Ser Pro

165 170 175

Ser Phe Thr Pro His Val Leu Thr Val Asn Ala Gly Glu Asp Val Thr

180 185 190

Met Lys Ile Met Thr Phe Ser Gln Gln Gly Ser Arg Ala Ile Cys Ile

195 200 205

Leu Ser Ala Asn Gly Pro Ile Ser Asn Val Thr Leu Arg Gln Ser Met

210 215 220

Thr Ser Gly Gly Thr Leu Thr Tyr Glu Gly His Phe Glu Ile Leu Ser

225 230 235 240

Leu Thr Gly Ser Phe Ile Pro Ser Glu Ser Gly Gly Thr Arg Ser Arg

245 250 255

Ala Gly Gly Met Ser Val Ser Leu Ala Gly Gln Asp Gly Arg Val Phe

260 265 270

Gly Gly Gly Leu Ala Gly Leu Phe Ile Ala Ala Gly Pro Val Gln Val

275 280 285

Met Val Gly Ser Phe Ile Ala Gly Gln Glu Glu Ser Gln Gln Gln Gln

290 295 300

Gln Gln Ile Lys Lys Gln Arg Arg Glu Arg Leu Gly Ile Pro Thr Thr

305 310 315 320

Thr Gln Ala Ser Asn Ile Ser Phe Gly Gly Ser Ala Glu Asp Pro Lys

325 330 335

Ala Arg Tyr Gly Leu Asn Lys Pro Val Val Ile Gln Pro Pro Pro Val

340 345 350

Ser Ala Pro Pro Val Ser Phe Ser His Glu Pro Ser Thr Asn Thr Val

355 360 365

His Gly Tyr Tyr Ala Asn Asn Thr Ala Asn His Ile Lys Asp Leu Phe

370 375 380

Ser Ser Leu Pro Gly Glu Asp Arg Glu Glu Asp Glu Asp Asp Leu Glu

385 390 395 400

Gly Glu Asp Asp Glu Glu Phe Gly Gly His Ser Glu Ser Asp Thr Glu

405 410 415

Val Pro Ser

Claims

1.AHL4The application of transcription factor in regulating and controlling plant lipid metabolism; the application is represented asAHL4Gene deletion promotes hydrolysis of TAG;AHL4gene overexpression inhibits TAG hydrolysis; the above-mentionedAHL4The gene is Arabidopsis thalianaAHL4The gene and the sequence are shown in SEQ ID NO. 2.

2. The method of claim 1AHL4The application of the transcription factor in regulating and controlling the metabolism of plant lipid is characterized in that:AHL4the gene deletion realizes the promotion of TAG hydrolysis by promoting the expression of TAG hydrolysis related genes and beta-oxidation related genes;AHL4the gene overexpression realizes the inhibition of TAG hydrolysis by inhibiting the expression of TAG hydrolysis related genes and beta-oxidation related genes; the TAG hydrolysis-related gene isSDP1AndDALL5the beta-oxidation related gene isKAT5。

3.AHL4The application of transcription factor in the establishment of plant seedling regulating system; the application is represented asAHL4Gene deletion promotes the germination rate and/or main root elongation of plant seeds;AHL4gene overexpression inhibits the germination rate and/or main root elongation of plant seeds; the above-mentionedAHL4The gene is Arabidopsis thalianaAHL4The gene and the sequence are shown in SEQ ID NO. 2.

4. Lifting deviceA method for increasing the oil content of a plant seed, comprising: comprising overexpression in plantsAHL4A gene; the above-mentionedAHL4The gene is Arabidopsis thalianaAHL4The gene and the sequence are shown in SEQ ID NO. 2.

5. The method of claim 4, wherein the overexpression is in the plantAHL4The method of the gene comprises: the primer sequences shown in SEQ ID NO.5 and SEQ ID NO.6 are utilized to amplify in an arabidopsis thaliana wild type DNA template to obtain a target fragment, then the target fragment is connected to a pBin35sRed1 vector in an enzyme digestion manner, and then the recombinant plasmid is converted into the arabidopsis thaliana wild type by adopting an agrobacterium dipping method.

6. A method for increasing the content of unsaturated fatty acids in plant seeds, characterized in that: comprising overexpression in plantsAHL4A gene; the above-mentionedAHL4The gene is Arabidopsis thalianaAHL4The gene and the sequence are shown in SEQ ID NO. 2.

7. The method of claim 6, wherein the unsaturated fatty acid content of the plant seed is increased by: overexpression in plantsAHL4The method of the gene comprises: the primer sequences shown in SEQ ID NO.5 and SEQ ID NO.6 are utilized to amplify in an arabidopsis thaliana wild type DNA template to obtain a target fragment, then the target fragment is connected to a pBin35sRed1 vector in an enzyme digestion manner, and then the recombinant plasmid is converted into the arabidopsis thaliana wild type by adopting an agrobacterium dipping method.